Fig 2: Effects of bile acid treatment on the mRNA and protein expression of mucins in pancreatic ductal cells. (A) Capan-1 and BxPC-3 cells were treated with different bile acids (BAs) for 24, 48 and 72 h and the relative gene expressions of MUC4 gene was investigated by real-time PCR. (B) Representative immunofluorescence staining of Capan-1 and BxPC-3 cells shows the expression of MUC4 after the treatment with taurochenodeoxycholic acid (TCDCA; 500 µM) for 24, 48 and 72 h. (C) Quantification of MUC4 protein expression. Specimens were scanned using Olympus IX83 based system (Olympus cellSense Dimension software version 2.3, https://www.olympus-lifescience.com/en/software/cellsens/) and DAB staining intensities were analysed by ImageJ software. Data represent mean ± SEM of three, independent experiments. * = p = 0.05 vs. Control. Scale bar represents 100 µm. (D) The cluster analysis and dendrogram show the difference between the effect of BAs treatment at different concentrations and time points. Red and white colours indicate high and low expression, respectively. (Values represent the fold change in the gene expression level of MUC genes). Data represent mean ± SEM of three, independent experiments. GCA glycocholic acid, TCA taurocholic acid, GDCA glycodeoxycholic acid, TDCA taurodeoxycholic acid, GCDCA glycochenodeoxycholic acid, TCDCA taurochenodeoxycholic acid.

Fig 3: Schematic diagram of the effect of bile acids. Bile acids (BAs) decrease the cell viability of normal pancreatic ductal cells and induce cell death in order to avoid malignant transformation. In the case of pancreatic ductal adenocarcinoma (PDAC) bile promotes the tumorigenic potential of the cancer cells in which the increased expression of MUC4 plays essential role.

Fig 4: Establishment and characterization of immortalized HAE depleted of MUC1. Immortalized airway epithelial BCi-NS1.1 cells were transduced with CRISPR/Cas9 and sgRNA targeting (A) MUC1 exon 5 for protein depletion (MUC1KO) or without (control [Ctl]) the predicted targeting site. (B) Genomic DNA was extracted and used in a T7 endonuclease I cleavage assay demonstrating editing at the target site. (C) After differentiation, total HAE lysate was collected, separated by PAGE, and blotted for nontargeted tethered mucin MUC4 (extracellular domain), MUC1-ED, MUC1-CT, and actin. (D) Representative histological sections of paraffin-embedded cultures show normal ciliated epithelium. H&E counterstain. Bar = 20 µm. (E) Fluorescent microparticles were applied apically to indicated cultures to determine mucociliary transport rate. MCC between culture types was analyzed by the Mann-Whitney U test (****, P < 0.0001).

Fig 5: Knockdown of MUC4 in Capan-1 cells. The expression levels of MUC4 was investigated by RT-PCR (A) and immunohistochemistry (B) in control cells and in cells treated with specific siRNA for MUC4. Orange indicates Capan-1 whereas red BcPC-3 cells. The rate of viability (C), proliferation (D) and adhesion (E) was determined at 24, 48 and 72 h, whereas migration (F) and colony forming (G) at 72 h. Data represent mean ± SEM of three, independent experiments. a = p = 0.05 vs. Control, b = p = 0.05 vs. MUC4 KD, c = p = 0.05 vs. MUC4 KD + TCDCA. TCDCA: taurochenodeoxycholic acid, KD: knock down.